A Facile Approach To Construct Hyaluronic Acid Shielding Non-viral Nano Gene Vector

Non-viral gene complexes fabricated by supramolecular assembly showed great potential in gene therapy due to the suitable nanometer size, controllable structure and excellent biocompatibility. Fabrication non-viral gene delivery system with excellent bio-stability, low cytotoxicity and high transfection is one of the key points.PEI is one of the most effective vectors due to its proton sponge effect. However, PEI/DNA polyplexes tend to aggregate in physiological condition and lead to rapid clearance from the circulation. The high toxicity and low gene expression in vivo limits its applications. Hyaluronic acid (HA), a natural polysaccharide, is the major constituent of the extracellular matrix (ECM). HA exhibits excellent biocompatibility and biodegradability. In this research, HA and thiolated HA (HA-SH) was added to PEI/DNA complexes. Due to the electrostatic interaction, the ternary polyplexes were constructed via supramolecular assembly. The relation between the structure of gene assembly and transfection efficiency was also explored.A facile approach for polymer gene carriers was used to construct hyaluronic acid (HA) shielding polyplexes. By adding HA to PEI/DNA complexes, theξ-potential of ternary polyplexes was changed from positive to negative. Spherical particles with diameter about 250 nm were observed by AFM and TEM. Ethidium bromide exclusion assay and gel retardation assay indicated that the electrostatic complexation was loosened after addition of HA. However, DNA disassembly did not occur. The proper reason was that the intensity of negative charges was not strong enough to release DNA from the complexes in our experiment. The stability of PEI/DNA/HA polyplexes in physiological salt condition was improved and the cytotoxicity was reduced. Comparing with PEI/DNA polyplexes, the uptake and transfection efficiency of HA shielding polyplexes was lower for HEK293T cells probably due to the reduced adsorptive endocytosis, whereas it was higher for HepG2 cells due to HA receptor mediated endocytosis. Proteins with negative charge are abundant in vivo, which probably lead to the disassembly of gene complexes. Caged polyplexes via biomimetic cross-linker were fabricated to improve the stability and transfection. Thiolated HA (HA-SH) was synthesized and then added to PEI/DNA complexes. In situ shell-cross-linking polyplexes were prepared by the oxidation of sulphydryl in aerial condition. Spherical particles with diameter about 200 nm were obtained. Comparing with PEI/DNA/HA polyplexes, the cross-linking shell showed more compact condensation with DNA. The stability in physiological condition was improved. Gel retardation assay indicated that at the existence of polyacrylic acid (PAA), PEI/DNA/HA polyplexes were disassembly and free DNA band was observed. However, the stability of PEI/DNA/HA-SH polyplexes was obviously improved with the increase of oxidation time. At GSH concentration of 3mmol/L, the disulfide bonds on the cross-linking shell was cleaved. Free DNA was released from the PEI/DNA/HA-SH polyplexes at the existence of PAA. The GSH-responsive polyplexes were successfully constructed. Comparing with PEI/DNA polyplexes, the uptake and transfection efficiency of shell cross-linking polyplexes was lower for HEK293T cells probably due to the reduced adsorptive endocytosis. By incubated cells with glutathione monoester (GSH-OEt), the concentration of GSH intracellular was effectively improved, followed by the improvement of the transfection efficiency. This facile approach to construct crosslinking polyplexes might have great potential application in non-viral gene delivery research.